Bioluminescence is the capacity of living organisms to emit visible light through a variety of chemiluminescent reaction systems. Bioluminescence reactions require three major components: a luciferin, a luciferase and molecular oxygen. However other components may also be required in some reactions, including cations (Ca++ and Mg++) and cofactors (ATP, NAD(P)H). Luciferases are enzymes that catalyse the oxidation of a substrate, luciferin, and produce an unstable intermediate. Light is emitted when the unstable intermediate decays to its ground state, generating oxyluciferin. There are many different unrelated types of luciferin, although many species from at least seven phyla use the same luciferin, known as coelenterazine, which contains a ring formed by three amino acids (2 tyrosines, and a phenylalanine). In some animals (e.g. jellyfish) the luciferin/luciferase system can be extracted in the form of a stable “photoprotein” which emits light upon calcium binding. Photoproteins differ from luciferases in that they are stabilized oxygenated intermediate complexes of luciferase and luciferin. Photoproteins are present in many marine coelenterates and allow these organisms to emit light for a variety of purposes including breeding, feeding, and defense (1). While bacteria emit light continuously, in many other organisms luminescence occurs as flashes, typically of 0.1-1 sec. duration. This requires a rapid switch on/off of the enzymatic reaction and the presence of reagents appropriately sequestered and ready to quick mobilization. In coelenterates, flashing is caused by calcium entry. The calcium binding sites of photoproteins are homologous to calmodulin. In the presence of calcium, photoproteins emit visible light through an intramolecular reaction. There are many luminescent organisms, but only seven photoproteins, namely Thalassicolin (2,3), Aequorin (4-6), Mitrocromin (syn. Halistaurin) (7,8), Clytin (syn. with Phialidin) (8,9) Obelin (2,6,10,11), Mnemiopsin (12,13), and Berovin (12,13), have been isolated so far. All these proteins are complexes of an apoprotein, an imidazopyrazine chromophore (coelenterazine), and oxygen. Their structures are highly conserved, especially in the region containing the three calcium binding sites (EF-hand structures). These EF-hand structures are characteristic of the calcium-binding protein family. The photoprotein emits light upon reaction with calcium which tightly binds to the EF-hand pocket. The reaction is a single turnover event and results in the release of CO2 and emission of light in the blue region (λmax=470 nm). The term “photoprotein” identifies the luciferin-bound polypeptide, which is capable of luminescence, while “apophotoprotein” is used to indicate the protein without luciferin.
The most studied photoproteins are Aequorin, isolated from Aequorea Victoria (14) and Obelin, isolated from Obelia longissima (15). Upon binding Ca++ Aequorin undergoes a conformational change converting itself into an oxygenase (luciferase), which then catalyzes the oxidation of coelenterazine by the bound molecular oxygen. The blue fluorescent protein is made up of coelenteramide, which is an oxidation product of coelenterazine, not covalently bound to apophotoprotein. The photoprotein may be regenerated from the apophotoprotein by incubation with coelenterazine, molecular oxygen, EDTA and 2-mercaptoethanol or dithiothreitol. Since coelenterazine is the common luminescent substrate used by the photoproteins Aequorin, Mitrocomin, Clytin and Obelin, the light-emitting reaction is likely the same in these four photoproteins (16). The recent acquisition of the primary structure and of the crystallographic data of Aequorin and Obelin gave rise to additional information on their function. Native Obelin from the hydroid Obelia longissima is a single-chain protein of 195 amino acid residues (aa) with an approximate molecular mass of 20 kDa that contains the noncovalently bound chromophoric group coelenterazine. The analysis of the primary structures of Clytin shows that it contains 189 aa and belongs to the family of photoproteins. The hydrozoan Ca++-binding photoprotein, however, differ from other Ca++-binding proteins such as calmodulin and troponin C by a relatively high content of cysteine, histidine, tryptophan, proline and tyrosine residues.
Studies of Obelin structure and function are reported in Bondar V S et al., Biochemistry (2001), 66(9):1014-8, Vysotski E S et al., (2003), 42(20): 6013-24 and Deng L. et al., FEBS Lett. (2001), 506(3): 281-5. The two latter, in particular, describe bioluminescence and emission properties of a W92F obelin mutant.
Photoproteins are widely used in reporter gene technology to monitor the cellular events associated with signal transduction and gene expression.
The study of cellular events and their regulation requires sensitive, non invasive analytic methods. Photoproteins and in general bioluminescence are excellent reporter systems as they have virtually no background in contrast to fluorescence systems.
Photoproteins have been expressed in mammalian cells to monitor calcium changes in response to different stimuli. Intracellular calcium concentration can be measured by adding coelenterazine cofactor to mammalian cells expressing the photoprotein and detecting photon emission, which is indicative of intracellular calcium concentration.